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DESCRIPTION:
Ready-to-use fluorometric substrate for caspase-9/Mch-6 and related caspases that recognize the amino acid sequence LEHD. Caspase activity can be quantified by fluorescent detection of free AFC after cleavage from the peptide substrate LEHD-AFC at Ex. = 400 nm and Em. = 505 nm, using a fluorometer or multi-well fluorescence plate reader. Alternatively, a shift in fluorescence from blue to green upon cleavage can be visualized, using a handheld long-UV lamp. The ready-to-use caspase substrate provides an economic alternative for researchers who perform large amount of caspase assays. Cell Lysis Buffer (Cat. #1067-100, -400) and 2X Reaction Buffer (Cat. #1068-20, -80) and DTT (Cat.# 1201-1) for caspase assays are also available separately.

ASSAY PROTOCOL:
1. Induce apoptosis in cells by desired method. Concurrently incubate a control culture without induction.
2. Count cells and pellet 1-5 x 106 cells or use 50-200 μg cell lysates if protein concentration has been measured.
3. Resuspend cells in 50 μl of chilled Cell Lysis Buffer (Cat.# 1067-100).
4. Incubate cells on ice for 10 minutes.
5. Add 50 μl of 2X Reaction Buffer (Cat.# 1068-20, -80) containing 10 mM DTT (Cat.#1201-1) to each sample.
6. Add 5 μl of the 1 mM LEHD-AFC (50 µM final conc.) into each tube individually and incubate at 37°C for 1-2 hour.
7. Read samples in a fluorometer equipped with a 400-nm excitation filter and 505-nm emission filter. For a plate-reading set-up, transfer the samples to a 96-well plate. You may perform the entire assay directly in a 96-well plate. Fold-increase in LEHD-dependent activity can be determined by comparing these results with the level of the uninduced control.